Binary transgene induction with distinct subtype-specific drivers permits marking and/or manipulation of different subsets of brain cells in intact organisms. However, one cannot differentially mark or independently manipulate distinct brain cells (e.g. specific neurons and their synaptic partners) in the same organism with only one binary transgene induction system. Here we propose to build tools for studying Drosophila neural circuit development with two noninterfering binary transcriptional systems (GAL4/UAS and LexA/lexAop). We will isolate diverse LexA drivers for targeting a large variety of different brain cells independently of GAL4/UAS. We will also generate various LexA-dependent transgenes for selectively marking or manipulating LexA-positive cells. These reagents would constitute what most Drosophila neurobiologists need to immediately apply the power of two independent binary transgene induction systems to their studies of neural circuit development. In addition, we will build new genetic mosaic toolkits on top of LexA/lexAop and without the GAL4 repressor GAL80. These GAL4/UAS/GAL80-independent genetic mosaic systems guarantee maximal versatility in the co-application of multiple genetic/transgenic tools. The simultaneous application of two independent binary transgene induction systems promises to further revolutionize modern neurobiological research by supporting complex genetic studies involving respective analysis or differential manipulation of distinct brain cells at the same time. This will allow more thorough elucidation and finer spatiotemporal manipulation of neural circuit development, and potentially lead to the identification of new therapeutic targets for remedying abnormal brain development or restoring neural circuitry in various neurodegenerative conditions. PUBLIC HEALTH RELEVANCE Wiring of neural circuits involves intricate cell-cell interactions among neurons and between neurons and glial cells that help govern individual growth cones of neurites to navigate, elaborate, and finally make synaptic contacts with specific targets. Many congenital malfunctions of the brain result from aberrant wiring of neural circuits. To understand how wiring of circuitry goes awry in such neurological disorders requires elucidation of the cellular and molecular bases of the diverse complex processes of cell-cell interactions. Furthermore, knowledge about neural circuit development may provide powerful clinical approaches for interventions during degenerative disorders or after injury. The goal of this project is to build tools for better studying cell-cell interactions in the highly convoluted central nervous system. We propose to develop such tools in the fruit fly Drosophila, a powerful model organism for understanding brain development and function. Binary transgene induction with distinct subtype-specific drivers permits marking and/or manipulation of different subsets of brain cells in intact organisms. However, one cannot differentially mark or independently manipulate distinct brain cells at the same time with only one binary transgene induction system. To independently target multiple neuron types or presynaptic versus postsynaptic cells or neurons versus glia, we propose to establish a second widely applicable binary transgene induction system in Drosophila. This involves generation of both subtype-specific drivers and a set of driver-dependent transgenes for marking or manipulating various specific Drosophila brain cells independently of the existing binary transcriptional system and all its derived genetic/transgenic tools. In addition, we will build more versatile genetic mosaic techniques on top of the new binary transgene induction system. The simultaneous application of two independent binary transgene induction systems promises to further revolutionize modern neurobiological research by supporting complex genetic studies involving respective analysis or differential manipulation of distinct brain cells in the same organism. Such studies will have fundamental impacts on our detailed mapping of neural circuitry, the elucidation and manipulation of neural circuit development, and the ultimate understanding of brain development and function. [unreadable] [unreadable]